Ex vivo anchored PD‐L1 functionally prevent in vivo renal allograft rejection

Abstract Organ transplantation is the optimal treatment for patients with end‐stage diseases. T cell activation is a major contributing factor toward the trigger of rejection. Induction therapy with T cell depleting agent is a common option but increases the risk of severe systemic infections. The ideal therapy should precisely target the allograft. Here, we developed a membrane‐anchored‐protein PD‐L1 (map‐PD‐L1), which effectively anchored onto the surface of rat glomerular endothelial cells (rgEC). The expression of PD‐L1 increased directly with map‐PD‐L1 concentration and incubation time. Moreover, map‐PD‐L1 was even stably anchored to rgEC at low temperature. Map‐PD‐L1 could bind to PD‐1 and significantly promote T cell apoptosis and inhibited T cell activation. Using kidney transplantation models, we found that ex vivo perfusion of donor kidneys with map‐PD‐L1 significantly protected grafts against acute injury without using any immunosuppressant. We found map‐PD‐L1 could reduce T cell graft infiltration and increase intragraft Treg infiltration, suggesting a long‐term effect in allograft protection. More importantly, modifying donor organs in vitro was not only safe, but also significantly reduced the side effects of systemic application. Our results suggested that ex vivo perfusion of donor organ with map‐PD‐L1 might provide a viable clinical option for organ‐targeted induction therapy in organ transplantation.

Programmed cell death protein-1 (PD-1) and its ligand PD-L1 are important immune checkpoint molecules that negatively regulate the immune system. 7,8 It has been reported that PD-L1 represses rejection in transplantation, and lack of PD-L1 has been shown to reduce survival time after islet transplantation. 9 In recent years, many research groups have attempted to upregulate PD-L1 expression in grafts through viral transfection and genetic engineering, which has produced a positive effect on the survival of grafts. 10,11 However, these methods are unable to achieve selective targeting, and the risk of systemic administration still hinders their further clinical application. Therefore, a fast, efficient and simple method to upregulate PD-L1 expression in organs will promote the application of PD-L1-based therapy in transplantation immunity.
Cell surface engineering is an effective technique for expressing new proteins on cell membranes without gene transfer. The key component of this technique is the membrane anchoring structure, which secures the target protein to the surface of the cell. 12,13 Through this, the target protein can be "implanted" on the cell surface, and the innate biological activity and function of the protein can be preserved. 14

| Design and synthesis of the map-PD-L1
The protein amino acid sequence of rat-derived PD-LI and anchoring structure APT542 were codon optimized using MaxCodonTM Optimization Program (V13) (DetaiBio). The PD-L1 + APT542 gene was inserted into the expression vector pcDNA3.1(À) by wholegenome synthesis and double enzyme digestion. Accuracy of the final expression vector was confirmed by restriction enzyme digestion and sequencing. Finally, the vector was transformed into DH5a cells, and the plasmid was extracted using a plasmid extraction kit.
The plasmid was then transfected into mammalian HEK293 cells for transient expression, and the fusion protein map-PD-L1 was purified using affinity chromatography. The expression and specificity of the synthesized protein was verified by Western blot. The membrane-localizing thiol-reactive agent APT542 was generated by solid-phase synthesis, and APT542 without PD-L1 was used as a control in this study.

| Cell line
Rat glomerular endothelial cells (rgEC) were kindly provided by Hui Peng (The Third Affiliated Hospital of Sun Yat-sen University). rgEC were incubated in RPMI-1640 supplemented with 10% FBS and 10% NuSerum (Sigma-Aldrich) in a cell incubator at 37 C under 5% CO 2 , as previously described. 15

| Immunofluorescence
To verify the ability of map-PD-L1 to anchor to the cell membrane, we incubated rgEC with 5 μg/ml map-PD-L1 or PD-L1 at 37 C for

| Flow cytometry
To explore the efficiency of map-PD-L1 anchoring to rgEC, a cell suspension was generated, consisting of 1 Â 10 5 per tube. Subsequently, cells were washed twice with PBS and then incubated with fluorescein isothiocyanate (FITC)-labeled map-PD-L1 at different concentrations, temperatures, and incubation times. The cells were then fixed in 4% paraformaldehyde (C01-06002; Bioss) and analyzed using a FACscan flow cytometer (Becton Dickinson).

| Coculture test
Referring to the above method, rgEC were cultured in a 24-well cell culture plate (Corning Inc.). When the cell density reached at least 50%, 50 μg/ml of map-PD-L1 were added to the experimental group and incubated for 30 min at 37 C. For the control group, an equal volume of medium was added instead. After incubation, cells were washed twice with PBS, and then preacquired Lewis rat spleen lymphocytes were cocultured with the rgEC at a density of 2 Â 10 6 cells/well.
For the T cell apoptosis assay, cells were cocultured for 3 days.
The supernatant was centrifuged to obtain mixed cultured lymphocytes, which were then washed twice with PBS and incubated with To analyze apoptosis of rgEC, cells were cocultured with anti-CD3 (1 μg/ml; ab16669; Abcam) and anti-CD28 (1 μg/ml; ab243228; Abcam) antibodies for 3 days as a stimulation protocol. After the supernatant was removed, the remaining adherent cells were washed twice with PBS and detected using a TUNEL Apoptosis Assay Kit (C1086; Beyotime). The rats were divided into the following groups: map-PD-L1 (anchored map-PD-L1), APT542 (APT542 anchoring) and control (simple perfusion solution). Donor kidney procurement and transplantation were performed under anesthesia with isoflurane as described previously. 16 Briefly, the left donor kidney of the BN rat was obtained, then Lewis rat was anesthetized. The aorta, renal vein, and ureter of the graft were anastomosed to the aorta, inferior vena cava, and ureter, respectively, of the recipient using 8-0 sutures. Both native kidneys were excised at the time of engraftment, and the total surgical ischemia time was restricted to less than 45 min. The control and blank groups were also treated by the process detailed above.

| Graft harvesting
To evaluate the transplanted kidney function of both the experimental and control groups, blood samples were collected from the groups before surgery, and 1, 3, and 5 days after surgery. The samples were centrifuged, and stored at a low temperature. Serum creatinine and blood urea nitrogen (BUN) levels were measured using the appropriate kits. During the follow-up, the survival time was recorded.

| Histologic analysis of allograft rejection
To assess the pathological changes and inflammatory cell infiltration of the transplanted kidney, the graft was obtained 5 days after F I G U R E 1 Study design. The recombinant protein map-PD-L1, which retains the function of PD-L1 and is anchored to the cell membrane, was expressed (see the yellow dashed box schematic). The ability to perform ex vivo perfusion is an important feature of the kidney. The kidneys were treated with a perfusion solution premixed with map-PD-L1, and map-PD-L1 was "implanted" on the surface of renal endothelial cells by incubation for the appropriate time. map-PD-L1, membrane-anchored-protein PD-L1 transplantation, then fixed with 4% paraformaldehyde and embedded with paraffin. Histologic sections for light microscopy were cut to a thickness of 3 μm, stained with hematoxylin and eosin (H&E) as well as Periodic acid-Schiff (PAS), and reviewed by an experienced pathologist who was blinded to the groups. Cell-mediated injuries included interstitial inflammation, tubulitis and acute tubular necrosis were scored by the semiquantitative Banff scoring criteria: 0, absent; 1, mild; 2, moderate; 3, prominent. 17,18 Immunofluorescence was used to detect the expression of PD-L1 (20 μg/ml; NBP1-76769; Novus Biologicals) in the grafts and the samples were examined using confocal laser scanning microscope (Zeiss LSM880).

| Quantitative real-time polymerase chain reaction
Total RNA was extracted from frozen kidney tissue using TRIzol reagent (Invitrogen) on homogenized samples. A total of 2 μg of RNA was reversed-transcribed using PrimeScript RT Master Mix

| Body weight and feed utilization
The recipients were observed daily, and body weight and feed consumption were recorded interval day. Feed utilization was determined using the following calculation: Feed utilization (%) = (body weight gain/feed consumption) Â 100.

| Statistical analysis
Normal distribution was first used to test the distribution of data using KS normality test. All data were normal distribution. Differences between groups were evaluated using either Student's t-test or analysis of variance using SPSS 19.0 software (SPSS Inc.). Graft survival among groups was compared using a log rank test. Data are shown as mean ± SD. A p < 0.05 was considered statistically significant.

| Study design and synthesis process and identification of map-PD-L1
The study design is as shown in Figure 1, in order to verify whether the extracellular domain of PD-L1 can be anchored and expressed in donor organs, and play a role in regulating immune response and preventing acute rejection. We used DNA sequencing to identify the expression vector pcDNA3.1(À) containing the PD-L1 + APT542 gene. Once the correct gene was obtained, a transfection-grade plasmid was generated and screened (Figure 2a).
The transfection plasmid was transfected into HEK293 cells for expression of the target protein, which was then purified using Ni-IDA affinity chromatography (Figure 2b), ultimately yielding the fusion protein map-PD-L1, at >90% purity. The protein amino acid sequence of map-PD-L is shown in Table S1. Western blot analysis was performed to confirm that the synthesized fusion protein was recognized by anti-His and anti-PD-L1 specific antibodies (Figure 2c,d).
In vitro experiments to verify the biological functions of map-PD-L1. Spleen lymphocytes were cocultured with rgEC. For the T cell activation assay, cells were cocultured with anti-CD3 and anti-CD28 antibodies for 1 day as a stimulation protocol. In the experimental group, map-PD-L1 was anchored to rgEC. Apoptosis (a) and activation (b) of T cells were analyzed by PI and annexin V staining, and CD69 molecular markers, respectively.

| Map-PD-L1 can bind to PD-1 and promote T cell apoptosis and inhibits T cell activation
A sandwich ELISA was used to detect the binding between the recombinant map-PD-L1 and PD-1 (schematic diagram of ELISA was shown in Figure S1A). Through the standard ELISA procedure and statistical analysis of data, we found that the map-PD-L1 with anchoring

| Map-PD-L1 can be locally anchored in the donor kidney
We sought to confirm that map-PD-L1 could be effectively anchored to the donor kidney during the perfusion phase in vitro.

| Map-PD-L1 improves graft function and prolongs graft survival in vivo
By constructing a rat kidney transplantation model, we confirmed that the survival time of the anchored map-PD-L1 group was significantly longer than that of both the APT542 and control groups (p < 0.05) (Figure 6a).
The serum creatinine and urea nitrogen levels in the map-PD-L1 group decreased compared with the other groups. Furthermore, significant differences were apparent in the serum creatinine level at 3 and 5 days postsurgery (p < 0.05) (Figure 6b). There was also a significant difference in BUN serum levels 5 days after surgery (p < 0.05) (Figure 6b).

| Map-PD-L1 alleviates pathological damage of acute renal allograft rejection
We used a kidney transplant model with a high degree of rejection after transplantation, with BN rats as donors and Lewis rats as recipients. The kidney specimens were obtained 5 days after operation. Using H&E and PAS staining (Figure 6c), we observed differences between the experimental and control groups in the degree of renal interstitial infiltration (Figure 6d), tubulitis (Figure 6e), and acute tubular necrosis (Figure 6f).

| Map-PD-L1 increases the infiltration of Treg cells in the allograft
Since the presence of Tregs can be beneficial for graft outcome, we investigated the effect of map-PD-L1 on recipient Treg cells. Kidney specimens were obtained 5 days after transplantation and RT-PCR was used to analyze Foxp3 mRNA levels, as Foxp3 is a marker of Tregs. We found an increase in Foxp3 mRNA levels in the map-PD-L1 group compared to the control groups ( Figure S3B). Foxp3+ Treg cells were visible in allograft of the group expressing anchored map-PD-L1, whereas in contrast, few Foxp3+ cells were observed in the control group (p < 0.05) (Figure 7d).

| Safety evaluation of map-PD-L1
To explore whether map-PD-L1 could be released into the blood circulation quickly after the kidney was reopened to the blood supply, causing the circulating level of PD-L1 to increase and causing systemic effects, we collected serum samples at 1, 3, and 5 days postsurgery, and used ELISA to detect the expression level of PD-L1. However, the level of PD-L1 was blow the lowest standard and the level of confident detection of the ELISA (data not shown). In addition, the mean body weight and feed utilization of the recipients are shown in Figure 8a,b, respectively. We found that the body weight of map-PD-L1-treated rats was significantly different from that of the control and APT542 groups on the fifth day after transplantation (p < 0.05), and there were significant differences in food utilization between the groups (p < 0.05). With the development of protein anchoring technology, researchers have begun to use anchors to express target proteins on the cell surface in an "implantable" way. 22,23 Studies have confirmed that anchoring of the extracellular domain of the target protein on the surface of the target cell, using structures such as APT542, results in increased activity compared to the free extracellular domain. 24,25 In this study, we found that map-PD-L1 could anchor to the surface of rgECs in a fast and efficient manner. In addition, our in vitro splenic lymphocyte coculture assay confirmed that map-PD-L1 could induce T cell apoptosis and inhibit T cell activation. Furthermore, we found that after anchoring map-PD-L1 on the surface of rgEC, they became resistant to the cell death effect mediated by mixed lymphocyte culture. We confirmed, for the first time, that the extracellular domain of PD-L1 retains its biological function after being anchored to the cell surface.

| DISCUSSION
Anchoring technology protects target proteins from being shed from the cell surface, thus prolonging their half-life. 26,27 The half-life of PD-L1 was about 18 h, 28 but we found that the experimental anchor group still had higher expression of PD-L1 than the other two groups on the fifth day after transplantation. This suggests that we were able to significantly prolong the half-life of PD-L1 by anchoring it to the cell surface. Although the local residence time of PD-L1 in the graft was only about 5 days, it is enough to reduce the incidence of acute rejection as the earliest several days after transplantation have the highest risk for dramatic rejection. This is why most induction therapies with high doses of immunosuppressive drugs were administered only within 3 days after surgery. 29 Therefore, we believe that the anchoring of map-PD-L1 in vitro holds promise as a feasible induction therapy.
In addition, we also determined that low temperature did not Data are expressed as mean ± SD. *p < 0.05, as determined using a Student's t-test. map-PD-L1, membrane-anchored-protein PD-L1 the main method of preservation. 30,31 We found that the anchoring of map-PD-L1 was easier to perform in a static storage environment.
Furthermore, this method did not increase the cost, and the operation was simple, indicating that this strategy could have a large clinical value.
Recent studies suggest that high expression of PD-L1 is the key to avoiding an immune attack and achieving immune tolerance after transplantation. 32,33 Therefore, many studies have focused on inducing high expression of PD-L1. 34,35 However, there is still no effective way to upregulate the local PD-L1 expression in target organs. In our study, by perfusing the donor kidney with map-PD-L1 prior to transplantation, we were able to induce high levels of expression of PD-L1 in a targeted manner. We found that the experimental group The mechanism of how PD-L1 affects T cells is clear. Numerous studies have confirmed that the interaction of PD-1 with PD-L1 could effectively drive T cell dysfunction. In this study, we found map-PD-L1 could promote T cell apoptosis and inhibit T cell activation, thereby reducing the infiltration of local T cells in the graft. Interestingly, we also found macrophage infiltration in the experimental group was significantly lower than that of the other groups. Moreover, the expression levels of macrophage-related inflammatory cytokines, such as TNF-α and IL-6, were also significantly downregulated, suggesting that, consistent with published research, 36 local high expression of PD-L1 also had an effect on macrophages. More importantly, we found that Foxp3+ cells appeared in the experimental group, but were hardly seen in the two control groups. Thus, after map-PD-L1 modification, T cells could be induced to differentiate into Treg cells, so as to further regulate the local immune response of the graft. Tregs have been shown to prolong allograft survival indefinitely without long-term systemic immunosuppression, and play a critical role in transplantation tolerance. 37,38 Some studies have illustrated the role of PD-1/PD-L1 axis in Treg development and function. 39 In our study, we further confirmed that, after anchoring on to the cell surface, the extracellular domain of PD-L1 could also regulate the differentiation of Treg cells, which might play a long-term role in inducing local immune tolerance. We also explored the effect of map-PD-L1 on humoral immunity, but the data were not specifically shown in the study because the differences between groups were not obvious, which indicated that map-PD-L1 worked by affecting T cells rather than B cells.
It should also be noted that our study was not the first to propose the protective effect of PD-L1 in the kidney. In Jaworska's reasech, 40 they found that both antibody neutralization and genetic deficiency of PD-L1 could accelerate kidney damage during ischemia reperfusion injury. However, the study did not explore the protective effect on the kidney from the aspect of overexpression of PD-L1 in the kidney. Preliminary safety analysis showed that map-PD-L1 did not increase the circulating concentration in vivo. We detected the circulating level of PD-L1 in each group at different time points, but the PD-L1 level did not achieve the lower limits of detection in the ELISA assay, suggesting that map-PD-L1 would not abruptly release from the donor kidney into the blood, which could cause some unpredictable side effects. We further analyzed the body weight and feed consumption in the recipient, and concluded that map-PD-L1 did not induce toxic side effects in recipients. These results provide a preliminary safety analysis for the further clinical transformation of map-PD-L1.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.